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The plant root microbiome consists of commensal, pathogenic and plant-beneficial microbes. Most members of the root microbiome possess microbe-associated molecular patterns (MAMPs) similar to those of plant pathogens. Their recognition by the cell surface-localized pattern recognition receptors can lead to the initiation of host immune signaling and suppression of plant growth due to growth-defense tradeoffs. We found that 42% of the tested root microbiota, including the plant growth-promoting rhizobacteria Pseudomonas capeferrum WCS358 (WCS358) and Pseudomonas simiae WCS417, are able to quench local Arabidopsis thaliana root immune responses that were triggered by the synthetic MAMP flg22, suggesting that this is an important function of the root microbiome. In a screen for WCS358 mutants that lost their capacity to suppress flg22-induced CYP71A12pro:GUS MAMP-reporter gene expression, we identified the bacterial genes pqqF and cyoB in WCS358. CyoB and pqqF are required for the production of gluconic acid and its derivative 2-keto gluconic acid. Both WCS358 mutants are impaired in the production of these organic acids and consequently lowered their extracellular pH to a lesser extent than wild-type WCS358. Acidification of the plant growth medium similarly suppressed flg22-induced CYP71A12pro:GUS and MYB51pro:GUS expression as well as the flg22-mediated oxidative burst, suggesting a role for rhizobacterial gluconic acid-mediated modulation of the extracellular pH in the suppression of local root immunity. Rhizosphere population densities of the mutants were significantly reduced compared to wild-type. Collectively, these findings show that suppression of local root immune responses is an important function of the root microbiome, as it facilitates colonization by beneficial root microbiota.

Significance Plant roots nurture a large diversity of soil microbes via exudation of chemical compounds into the rhizosphere. In turn, beneficial root microbiota promote plant growth and immunity. The root-specific transcription factor MYB72 has emerged as a central regulator in this process. Here, we show that MYB72 regulates the excretion of the coumarin scopoletin, an iron-mobilizing phenolic compound with selective antimicrobial activity that shapes the root-associated microbial community. Selected soil-borne fungal pathogens appeared to be highly sensitive to the antimicrobial activity of scopoletin, while two MYB72-inducing beneficial rhizobacteria were tolerant. Our results suggest that probiotic root-associated microbes that activate the iron-deficiency response during colonization stimulate MYB72-dependent excretion of scopoletin, thereby potentially improving their niche establishment and enhancing plant growth and protection., Plant roots nurture a tremendous diversity of microbes via exudation of photosynthetically fixed carbon sources. In turn, probiotic members of the root microbiome promote plant growth and protect the host plant against pathogens and pests. In the Arabidopsis thaliana–Pseudomonas simiae WCS417 model system the root-specific transcription factor MYB72 and the MYB72-controlled β-glucosidase BGLU42 emerged as important regulators of beneficial rhizobacteria-induced systemic resistance (ISR) and iron-uptake responses. MYB72 regulates the biosynthesis of iron-mobilizing fluorescent phenolic compounds, after which BGLU42 activity is required for their excretion into the rhizosphere. Metabolite fingerprinting revealed the antimicrobial coumarin scopoletin as a dominant metabolite that is produced in the roots and excreted into the rhizosphere in a MYB72- and BGLU42-dependent manner. Shotgun-metagenome sequencing of root-associated microbiota of Col-0, myb72, and the scopoletin biosynthesis mutant f6′h1 showed that scopoletin selectively impacts the assembly of the microbial community in the rhizosphere. We show that scopoletin selectively inhibits the soil-borne fungal pathogens Fusarium oxysporum and Verticillium dahliae, while the growth-promoting and ISR-inducing rhizobacteria P. simiae WCS417 and Pseudomonas capeferrum WCS358 are highly tolerant of the antimicrobial effect of scopoletin. Collectively, our results demonstrate a role for coumarins in microbiome assembly and point to a scenario in which plants and probiotic rhizobacteria join forces to trigger MYB72/BGLU42-dependent scopolin production and scopoletin excretion, resulting in improved niche establishment for the microbial partner and growth and immunity benefits for the host plant.

Disease suppressive soils typically develop after a disease outbreak due to the subsequent assembly of protective microbiota in the rhizosphere. The role of the plant immune system in the assemblage of a protective rhizosphere microbiome is largely unknown. In this study, we demonstrate that Arabidopsis thaliana specifically promotes three bacterial species in the rhizosphere upon foliar defense activation by the downy mildew pathogen Hyaloperonospora arabidopsidis. The promoted bacteria were isolated and found to interact synergistically in biofilm formation in vitro. Although separately these bacteria did not affect the plant significantly, together they induced systemic resistance against downy mildew and promoted growth of the plant. Moreover, we show that the soil-mediated legacy of a primary population of downy mildew infected plants confers enhanced protection against this pathogen in a second population of plants growing in the same soil. Together our results indicate that plants can adjust their root microbiome upon pathogen infection and specifically recruit a group of disease resistance-inducing and growth-promoting beneficial microbes, therewith potentially maximizing the chance of survival of their offspring that will grow in the same soil.

Trichoderma species and fluorescent Pseudomonas spp. have been reported to induce systemic resistance in plants. In this study the effectiveness of a combination of these biological control agents on the efficacy of induced resis- tance was investigated in cucumber and the model plant Arabidopsis thaliana. Trichoderma harzianum Tr6, and Pseudomonas sp. Ps14, both isolated from the rhizosphere of cucumber, were tested as a single application and in combination for their abilities to elicit induced resistance in cucumber against Fusarium oxysporum f. sp. radicis cucumerinum and in A. thaliana against Botrytis cinerea. The combination of Tr6 and Ps14 induced a significantly higher level of resistance in cucumber, which was associated with the primed expression of a set of defense-related genes upon challenge with Fusarium. In Arabidopsis both Ps14 and Tr6 triggered ISR against B. cinerea but their combination did not show enhanced effects. In the induced systemic resistance-defective Arabidopsis mutant myb72, none of the treatments protected against B. cinerea, whereas in the SA-impaired mutant sid2 all treatments were effective. Taken together, these results indicate that in Arabidopsis Ps14 and Tr6 activate the same signaling pathway and thus have no enhanced effect in combination. The enhanced protection in cucumber by the combi- nation is most likely due to activation of different signaling pathways by the two biocontrol agents.

The rhizosphere is the infection court where soil-borne pathogens establish a parasitic relationship with the plant. To infect root tissue, pathogens have to compete with members of the rhizosphere microbiome for available nutrients and microsites. In disease-suppressive soils, pathogens are strongly restricted in growth by the activities of specific rhizosphere microorganisms. Here, we sequenced metagenomic DNA and RNA of the rhizosphere microbiome of sugar beet seedlings grown in a soil suppressive to the fungal pathogen Rhizoctonia solani. rRNA-based analyses showed that Oxalobacteraceae, Burkholderiaceae, Sphingobacteriaceae and Sphingomonadaceae were significantly more abundant in the rhizosphere upon fungal invasion. Metatranscriptomics revealed that stress-related genes (ppGpp metabolism and oxidative stress) were upregulated in these bacterial families. We postulate that the invading pathogenic fungus induces, directly or via the plant, stress responses in the rhizobacterial community that lead to shifts in microbiome composition and to activation of antagonistic traits that restrict pathogen infection.

The plant growth-promoting rhizobacterium Pseudomonas fluorescens WCS374r produces several iron-regulated metabolites, including the fluorescent siderophore pseudobactin (Psb374), salicylic acid (SA), and pseudomonine (Psm), a siderophore that contains a SA moiety. After purification of Psb374 from culture supernatant of WCS374r, its structure was determined following isoelectrofocusing and tandem mass spectrometry, and found to be identical to the fluorescent siderophore produced by P. fluorescens ATCC 13525. To study the role of SA and Psm production in colonization of Arabidopsis thaliana roots and in induced systemic resistance (ISR) against Pseudomonas syringae pv. tomato (Pst) by strain WCS374r, mutants disrupted in the production of these metabolites were obtained by homologous recombination. These mutants were further subjected to transposon Tn5 mutagenesis to generate mutants also deficient in Psb374 production. The mutants behaved similar to the wild type in both their Arabidopsis rhizosphere-colonizing capacity and their ability to elicit ISR against Pst. We conclude that Psb374, SA, and Psm production by P. fluorescens WCS374r are not required for eliciting ISR in Arabidopsis.

Pseudomonas fluorescens strains that produce the polyketide antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) are among the most effective rhizobacteria that suppress root and crown rots, wilts, and damping-off diseases of a variety of crops, and they play a key role in the natural suppressiveness of some soils to certain soilborne pathogens. Root colonization by 2,4-DAPG-producing P. fluorescens strains Pf-5 (genotype A), Q2-87 (genotype B), Q8r1-96 (genotype D), and HT5-1 (genotype N) produced induced systemic resistance (ISR) in Arabidopsis thaliana accession Col-0 against bacterial speck caused by P. syringae pv. tomato. The ISR-eliciting activity of the four bacterial genotypes was similar, and all genotypes were equivalent in activity to the well-characterized strain P. fluorescens WCS417r. The 2,4-DAPG biosynthetic locus consists of the genes phlHGF and phlACBDE. phlD or phlBC mutants of Q2-87 (2,4-DAPG minus) were significantly reduced in ISR activity, and genetic complementation of the mutants restored ISR activity back to wild-type levels. A phlF regulatory mutant (overproducer of 2,4-DAPG) had ISR activity equivalent to the wild-type Q2-87. Introduction of DAPG into soil at concentrations of 10 to 250 μM 4 days before challenge inoculation induced resistance equivalent to or better than the bacteria. Strain Q2-87 induced resistance on transgenic NahG plants but not on npr1-1, jar1, and etr1 Arabidopsis mutants. These results indicate that the antibiotic 2,4-DAPG is a major determinant of ISR in 2,4-DAPG-producing P. fluorescens, that the genotype of the strain does not affect its ISR activity, and that the activity induced by these bacteria operates through the ethylene- and jasmonic acid-dependent signal transduction pathway.

Summary Pseudomonas putida KT2440 is an efficient colonizer of the rhizosphere of plants of agronomical and basic interest. We have demonstrated that KT2440 can protect the model plant Arabidopsis thaliana against infection by the phytopathogen Pseudomonas syrin- gae pv. tomato DC3000. P. putida extracellular haem- peroxidase (PP2561) was found to be important for competitive colonization and essential for the induc- tion of plant systemic resistance. Root exudates of plants elicited by KT2440 exhibited distinct patterns of metabolites compared with those of non-elicited plants. The levels of some of these compounds were dramatically reduced in axenic plants or plants colo- nized by a mutant defective in PP2561, which has increased sensitiveness to oxidative stress with respect to the wild type. Thus high-level oxidative stress resistance is a bacterial driving force in the rhizosphere for efficient colonization and to induce systemic resistance. These results provide important new insight into the complex events that occur in order for plants to attain resistance against foliar pathogens.

Ran, L. X., van Loon, L. C., and Bakker, P. A. H. M. 2005. No role for bacterially produced salicylic acid in rhizobacterial induction of systemic resistance in Arabidopsis. Phytopathology 95:1349-1355. The role of bacterially produced salicylic acid (SA) in the induction of systemic resistance in plants by rhizobacteria is far from clear. The strong SA producer Pseudomonas fluorescens WCS374r induces resistance in radish but not in Arabidopsis thaliana, whereas application of SA leads to induction of resistance in both plant species. In this study, we compared P. fluorescens WCS374r with three other SA-producing fluorescent Pseudomonas strains, P. fluorescens WCS417r and CHA0r, and P. aeruginosa 7NSK2 for their abilities to produce SA under different growth conditions and to induce systemic resistance in A. thaliana against bacterial speck, caused by P. syringae pv. tomato. All strains produced SA in vitro, varying from 5 fg cell –1 for WCS417r to >25 fg cell –1 for WCS374r. Addition of 200 µM FeCl3 to standard succinate medium abolished SA production in all strains. Whereas the incubation temperature did not affect SA production by WCS417r and 7NSK2, strains WCS374r and CHA0r produced more SA when grown at 33 instead of 28°C. WCS417r, CHA0r, and 7NSK2 induced systemic resistance apparently associated with their ability to produce SA, but WCS374r did not. Conversely, a mutant of 7NSK2 unable to produce SA still triggered induced systemic resistance (ISR). The possible involvement of SA in the induction of resistance was evaluated using SA-nonaccumulating transgenic NahG plants. Strains WCS417r, CHA0r, and 7NSK2 induced resistance in NahG Arabidopsis. Also, WCS374r, when grown at 33 or 36°C, triggered ISR in these plants, but not in ethylene-insensitive ein2 or in non-plant pathogenesis-related protein-expressing npr1 mutant plants, irrespective of the growth temperature of the bacteria. These results demonstrate that, whereas WCS374r can be manipulated to trigger ISR in Arabidopsis, SA is not the primary determinant for the induction of systemic resistance against bacterial speck disease by this bacterium. Also, for the other SAproducing strains used in this study, bacterial determinants other than SA must be responsible for inducing resistance.

The ability of selected strains of fluorescent Pseudomonas spp. to cause induced systemic resistance (ISR) in Eucalyptus urophylla against bacterial wilt caused by Ralstonia solanacearum was investigated. Four of the five strains used can produce salicylic acid (SA) in vitro and, therefore, chemical SA, that is known to induce resistance in many plant species, was used as a reference treatment. Whereas a soil drench with SA did induce systemic resistance in E. urophylla, infiltration of SA into leaves did not. None of the fluorescent Pseudomonas spp. strains caused ISR against bacterial wilt when applied to the soil, but two strains, P. putida WCS358r and P. fluorescens WCS374r triggered ISR when infiltrated into two lower leaves 3–7 days before challenge inoculation. A mutant of strain WCS358r defective in the biosynthesis of the fluorescent siderophore pseudobactin, did not cause ISR, while the purified siderophore of WCS358r did, suggesting that pseudobactin358 is the ISR determinant of WCS358. A siderophore-minus mutant of WCS374r induced the same level of disease resistance as its parental strain, but the purified siderophore induced resistance as well, indicating that both the siderophore and another, unknown, inducing determinant(s) of WCS374r can trigger ISR in Eucalyptus. A possible role of WCS374r-produced SA remains uncertain. Transformation of a siderophore-minus mutant of WCS358 with the SA biosynthetic gene cluster from WCS374 did not enable this transformant to cause ISR in E. urophylla.

Transgenic tobacco plants (Tetr) expressing the mutant etr1-1 gene from Arabidopsis thaliana are insensitive to ethylene and develop symptoms of wilting and stem rot when grown in nonautoclaved soil. Several isolates of Fusarium, Thielaviopsis, and Pythium were recovered from stems of diseased Tetr plants. Inoculation with each of these isolates of 6-week-old plants growing in autoclaved soil caused disease in Tetr plants but not in nontransformed plants. Also, when 2-week-old seedlings were used, nontransformed tobacco appeared nonsusceptible to the Fusarium isolates, whereas Tetr seedlings did develop disease. Tetr seedlings were not susceptible to several nonhost Fusarium isolates. In contrast to results with Fusarium isolates, inoculation of 2-week-old seedlings with a Thielaviopsis isolate resulted in equal symptom development of nontransformed and Tetr tobacco. In order to explore the potential range of pathogens to which Tetr tobacco plants display enhanced susceptibility, the pathogenicity of several root and leaf pathogens was tested. Tetr plants were more susceptible to the necrotrophic fungi Botrytis cinerea and Cercospora nicotianae and the bacterium Erwinia carotovora, but only marginally more to the bacterium Ralstonia solanacearum. In contrast, the biotrophic fungus Oidium neolycopersici, the oomycete Peronospora tabacina, and Tobacco mosaic virus caused similar or less severe symptoms on Tetr plants than on nontransformed plants. Total peroxidase activity of Tetr plants was lower than that of nontransformed plants, suggesting a role for peroxidases in resistance against necrotrophic microorganisms. A comparable range of pathogens was examined on Arabidopsis and its ethylene-insensitive mutants etr1-1 and ein2-1. With the exception of one Fusarium isolate, ethylene insensitivity increased susceptibility of Arabidopsis plants to a similar spectrum of necrotizing pathogens as in tobacco. Thus, both ethylene-insensitive tobacco and Arabidopsis plants appear to be impaired in their resistance to necrotrophic pathogens.

Pseudomonas putida WCS358r, genetically modified to have improved activity against soil-borne pathogens, was released into the rhizosphere of wheat. Two genetically modified derivatives carried the phz or the phl biosynthetic gene loci and constitutively produced either the antifungal compound phenazine-1-carboxylic acid (PCA) or the antifungal and antibacterial compound 2,4-diacetylphloroglucinol (DAPG). In 1997 and 1998, effects of single introductions of PCA producing derivatives on the indigenous microflora were studied. A transient shift in the composition of the total fungal microflora, determined by amplified ribosomal DNA restiction analysis (ARDRA), was detected. Starting in 1999, effects of repeated introduction of genetically modified microorganisms (GMMs) were studied. Wheat seeds coated with the PCA producer, the DAPG producer, a mixture of the PCA and DAPG producers, or WCS358r, were sown and the densities, composition and activities of the rhizosphere microbial populations were measured. All introduced strains decreased from 10(7) CFU per gram of rhizosphere sample to below the detection limit after harvest of the wheat plants. The phz genes were stably maintained in the PCA producers, and PCA was detected in rhizosphere extracts of plants treated with this strain or with the mixture of the PCA and DAPG producers. The phl genes were also stably maintained in the DAPG producing derivative of WCS358r. Effects of the genetically modified bacteria on the rhizosphere fungi and bacteria were analyzed by using amplified ribosomal DNA restriction analysis. Introduction of the genetically modified bacterial strains caused a transient change in the composition of the rhizosphere microflora. However, introduction of the GMMs did not affect the several soil microbial activities that were investigated in this study.

Fusarium wilt diseases, caused by the fungus Fusarium oxysporum, lead to significant yield losses of crops. One strategy to control fusarium wilt is the use of antagonistic, root-colonizing Pseudomonas spp. It has been demonstrated that different strains of these bacteria suppress disease by different mechanisms. Therefore, application of a mixture of these biocontrol strains, and thus of several suppressive mechanisms, may represent a viable control strategy. A prerequisite for biocontrol by combinations of biocontrol agents can be the compatibility of the co-inoculated micro-organisms. Hence, compatibility between several Pseudomonasspp. strains, that have the ability to suppress fusarium wilt of radish, was tested in vitro on KB agar plates. Growth of P. fluorescens strain RS111 was strongly inhibited by Pseudomonas spp. strains RE8, RS13, RS56 and RS158, whereas a mutant of strain RS111 (RS111-a) was insensitive to inhibition by these strains. Strains RS111 and RS111-a only slightly inhibited some other strains. Suppression of fusarium wilt of radish in a potting soil bioassay by the incompatible combination of RE8 and RS111 was comparable to the effects of the single strains. However, disease suppression by the compatible combination of RE8 and RS111-a was significantly better as compared to the single strains. In contrast, the incompatible combination of RS56 with RS111 resulted in enhanced disease suppression as compared to the single strains. Increased disease suppression by combinations of RS13 or RS158 with RS111 or RS111-a was not observed. This indicates that specific interactions between biocontrol strains influence disease suppression by combinations of these strains.

▪ Abstract Nonpathogenic rhizobacteria can induce a systemic resistance in plants that is phenotypically similar to pathogen-induced systemic acquired resistance (SAR). Rhizobacteria-mediated induced systemic resistance (ISR) has been demonstrated against fungi, bacteria, and viruses in Arabidopsis, bean, carnation, cucumber, radish, tobacco, and tomato under conditions in which the inducing bacteria and the challenging pathogen remained spatially separated. Bacterial strains differ in their ability to induce resistance in different plant species, and plants show variation in the expression of ISR upon induction by specific bacterial strains. Bacterial determinants of ISR include lipopolysaccharides, siderophores, and salicylic acid (SA). Whereas some of the rhizobacteria induce resistance through the SA-dependent SAR pathway, others do not and require jasmonic acid and ethylene perception by the plant for ISR to develop. No consistent host plant alterations are associated with the induced state, but upon challenge inoculation, resistance responses are accelerated and enhanced. ISR is effective under field conditions and offers a natural mechanism for biological control of plant disease.

Summary-.Dispersal of wild-type and genetically-modified strains of plant growth-promoting fluorescent Pseudomonas spp to aerial parts of radish plants was investigated using different inoculation strategies. P. putida strain WCS358rr colonized the surface of cotyledons of radish plants when seeds were coated with this strain or when non-coated seeds were sown in soil treated with WCS358rr. The cotyledons were not colonized by WCS358rr when suspensions of this strain were inoculated onto roots of radish seedlings that had already grown for 4 d in soil. Strain WCS358rr not only colonized the surface, but also the interior of cotyledons. For all types of inoculation strategies tested, strain WCS358rr colonized the root surface but did not penetrate the interior of the root. These results indicate that WCS358rr was not transported up, or downwards through the vascular system. When coated on seed, dispersal to cotyledons of radish was also demonstrated for P.fluorescens WCS374 and its genetically-modified derivative WCS374pMR. Plasmid pMR was maintained in WCS374pMR throughout the experiment. No differences were observed between modified strain WCS374pMR and wild-type strain WCS374 with regard to dispersal to and colonization of cotyledons and roots of radish. Strains WCS358rr, WCS374 and WCS374pMR survived on cotyledons for a period of 21-31 d. Dispersal of the applied bacterial strains to the first true leaves of radish could not be demonstrated. The results indicate that cotyledons of radish become contaminated with strains of Pseudomonas spp during germination of treated seeds or during seedling emergence from treated soil. The implications of dispersal of wild-type and genetically-modified pseudomonads to aerial parts of the plant are discussed.

Pseudomonas fluorescens-mediated induction of systemic resistance in radish against fusarium wilt (Fusarium oxysporum f. sp.raphani) was studied in a newly developed bioassay using a rockwool system. In this bioassay the pathogen and bacterium were confirmed to be confined to spatially separate locations on the plant root, throughout the experiment. Pathogen inoculum obtained by mixing peat with microconidia and subsequent incubation for four days at 22 °C, yielded a better percentage of diseased plants than a microconidial suspension drench, an injection of a microconidial suspension into the hypocotyl, or a talcum inoculum.Pseudomonas fluorescens strain WCS374 applied in talcum or peat, but not as a suspension drench, induced systemic resistance. A minimal initial bacterial inoculum density of ≥105 CFU WCS374 root−1 was required to significantly reduce the percentage diseased plants. At least one day was necessary between bacterization of strain WCS374 in talcum on the root tips and inoculation of the pathogen in peat on the root base, for an optimal induction of systemic resistance. Strain WCS374 induced systemic resistance in six radish cultivars differing in their susceptibility toF. oxysporum f. sp.raphani. Significant suppression of disease by bacterial treatments was generally observed when disease incidence in the control treatment, depending on pathogen inoculum density, ranged between approximately 40 to 80%. Strains WCS374 and WCS417 ofPseudomonas fluorescens induced systemic resistance against fusarium wilt, whereasP. putida WCS358 did not. This suggests that the induction of systemic resistance byPseudomonas spp. is dependent on strain-specific traits.

In this study, the potential of different Pseudomonas strains to utilize heterologous siderophores was compared with their competitiveness in the rhizosphere of radish. This issue was investigated in interactions between Pseudomonas putida WCS358 and Pseudomonas fluoresceins WCS374 and in interactions between strain WCS358 and eight indigenous Pseudomonas strains capable of utilizing pseudobactin 358. During four successive plant growth cycles of radish, strain WCS358 significantly reduced rhizosphere population densities of the wild-type strain WCS374 by up to 30 times, whereas derivative strain WCS374(pMR), harboring the siderophore receptor PupA for ferric pseudobactin 358, maintained its population density. Studies involving interactions between strain WCS358 and eight different indigenous Pseudomonas strains demonstrated that despite the ability of these indigenous isolates to utilize pseudobactin 358, their rhizosphere population densities were significantly reduced by strain WCS358 by up to 20 times. Moreover, rhizosphere colonization by WCS358 was not affected by any of these indigenous strains, even though siderophore-mediated growth inhibition of WCS358 by a majority of these strains was demonstrated in a plate bioassay. In conclusion, it can be stated that siderophore-mediated competition for iron is a major determinant in interactions between WCS358 and WCS374 in the rhizosphere. Moreover, our findings support the common assumption that cloning of siderophore receptor genes from one Pseudomonas strain into another can confer a competitive advantage in interactions in the rhizosphere. Interactions between WCS358 and the selected indigenous rhizosphere isolates, however, indicate that other traits also contribute to the rhizosphere competence of fluorescent Pseudomonas spp.Key words: siderophore, siderophore receptors, root colonization, fluorescent Pseudomonas.

The mechanisms of suppression of fusarium wilt of carnation by two fluorescentPseudomonas strains were studied.

Crop specificity of rhizosphere-inhabiting fluorescent Pseudomonas spp was studied for potato, grass and wheat grown in a silty loam soil. For each crop about 50 dominant Pseudomonas isolates were obtained from the rhizosphere. Isolates were distinguished by comparing their lipopolysaccharide (LPS) and cell envelope protein (CEP) patterns. About 30 distinct patterns were detected for each crop. The majority of LPS and CEP patterns observed for each crop was not detected on the other crops, suggesting a crop specificity for certain Pseudomonas populations. From each crop distinct Pseudomonas isolates were used to study crop-specific root colonization by growing potato, grass and wheat plants in soil bacterized with a mixture of these isolates of the three crops. Crop specificity in root colonization could not be demonstrated. The involvement of root agglutinins in the composition of rhizosphere Pseudomonas populations was examined. Reactions of distinct Pseudomonas isolates from each crop with crude or partially-purified agglutinin preparations of potato, grass or wheat did not correlate. We concluded that the composition of rhizosphere Pseudomonas populations differed between crops. However, crop-specific root colonization or crop-specific agglutination could not be demonstrated with the experimental approach used.

SUMMARY Pseudomonas putida WCS358 is a plant growth-promoting rhizobacterium originally isolated from the rhizosphere of potato. It can suppress soil-borne plant diseases by siderophore-mediated competition for iron, but it has also been reported to result in induced systemic resistance (ISR) in Arabidopsis thaliana . Bacterial determinants of this strain involved in inducing systemic resistance in Arabidopsis were investigated using a Tn 5 transposon mutant defective in biosynthesis of the fluorescent siderophore pseudobactin, a non-motile Tn 5 mutant lacking flagella, and a spontaneous phage-resistant mutant lacking the O-antigenic side chain of the lipopolysaccharides (LPS). When using Pseudomonas syringae pv. tomato as the challenging pathogen, purified pseudobactin, flagella and LPS all triggered ISR. However, the mutants were all as effective as the parental strain, suggesting redundancy in ISR-triggering traits in WCS358. The Botrytis cinerea ‐ tomato, B. cinerea ‐bean and Colletotrichum lindemuthianum ‐bean model systems were used to test further the potential of P. putida WCS358 to induce ISR. Strain WCS358 significantly reduced disease development in all three systems, indicating that also on tomato and bean WCS358 can trigger ISR. In both tomato and bean, the LPS mutant had lost the ability to induce resistance, whereas the flagella mutant was still effective. In bean, the pseudobactin mutant was still effective, whereas this mutant has lost its effectivity in tomato. In both bean and tomato, flagella isolated from the parental strain were not effective, whereas LPS or pseudobactin did induce systemic resistance.

The stability of rifampicin resistance in plant growth-promoting Pseudomonas putida strain WCS358 was studied in potato rhizosphere in the field. Three out of seven rifampicin-resistant mutants of strain WCS358 were selected in this study. Their specific growth rate, competitive growth in liquid medium and colonization of potato roots in non-sterile soil, was comparable to that of their parental strain. These rifampicin-resistant mutants were used to treat potato seed tubers, which were thereafter sown in the field. To test the stability of the rifampicin resistance in the field, about 1200 fluorescent Pseudomonas isolates obtained from underground plant parts at 82, 95, 109 and 130 days after seeding, were tested for rifampicin resistance and for agglutination with an antiserum specific for strain WCS358. Ail fluorescent Pseudomonas isolates that showed a positive agglutination reaction with the antiserum, were also rifampicin-resistant. Twelve agglutination-positive isolates, selected at random, were all identified as strain WCS358 from patterns of lipopolysaccharides after sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Thus, rifampicin resistance seems to be a stable marker in the mutants of strain WCS358 tested, also under field conditions. It is concluded that rifampicin resistance can be used as a reliable marker for ecological studies on rhizosphere pseudomonads.

Fluorescent Pseudomonas spp. have been studied for decades for their plant growth-promoting effects through effective suppression of soilborne plant diseases. The modes of action that play a role in disease suppression by these bacteria include siderophore-mediated competition for iron, antibiosis, production of lytic enzymes, and induced systemic resistance (ISR). The involvement of ISR is typically studied in systems in which the Pseudomonas bacteria and the pathogen are inoculated and remain spatially separated on the plant, e.g., the bacteria on the root and the pathogen on the leaf, or by use of split root systems. Since no direct interactions are possible between the two populations, suppression of disease development has to be plant-mediated. In this review, bacterial traits involved in Pseudomonas-mediated ISR will be discussed.

Biological control of soilborne plant pathogens in the field has given variable results. By combining specific strains of microorganisms, multiple traits antagonizing the pathogen can be combined and this may result in a higher level of protection. Pseudomonas putida WCS358 suppresses Fusarium wilt of radish by effectively competing for iron through the production of its pseudobactin siderophore. However, in some bioassays pseudobactin-negative mutants of WCS358 also suppressed disease to the same extent as WCS358, suggesting that an, as yet unknown, additional mechanism may be operative in this strain. P. putida strain RE8 induced systemic resistance against fusarium wilt. When WCS358 and RE8 were mixed through soil together, disease suppression was significantly enhanced to approximately 50% as compared to the 30% reduction for the single strain treatments. Moreover, when one strain failed to suppress disease in the single application, the combination still resulted in disease control. The enhanced disease suppression by the combination of P. putida strains WCS358 and RE8 is most likely the result of the combination of their different disease-suppressive mechanisms. These results demonstrate that combining biocontrol strains can lead to more effective, or at least, more reliable biocontrol of fusarium wilt of radish.

23 pages, Specific strains of fluorescent Pseudomonas spp. inhabit the environment surrounding plant roots and some even the root interior. Introducing such bacterial strains to plant roots can lead to increased plant growth, usually due to suppression of plant pathogenic microorganisms. We review the modes of action and traits of these beneficial Pseudomonas bacteria involved in disease suppression. The complex regulation of biological control traits in relation to the functioning in the root environment is discussed. Understanding the complexity of the interactions is instrumental in the exploitation of beneficial Pseudomonas spp. in controlling plant diseases.

A long-term field experiment (1999-2002) was conducted to monitor effects on the indigenous microflora of Pseudomonas putida WCS358r and two transgenic derivatives constitutively producing phenazine-1-carboxylic acid (PCA) or 2,4-diacetylphloroglucinol (DAPG). The strains were introduced as seed coating on wheat into the same field plots each year. Rhizosphere populations of ascomycetes were analysed using denaturing gradient gel electrophoresis (DGGE). To evaluate the significance of changes caused by the genetically modified microorganisms (GMMs), they were compared with effects caused by a crop rotation from wheat to potato. In the first year, only the combination of both GMMs caused a significant shift in the ascomycete community. After the repeated introductions this effect was no longer evident. However, cropping potato significantly affected the ascomycete community. This effect persisted into the next year when wheat was grown. Clone libraries were constructed from samples taken in 1999 and 2000, and sequence analysis indicated ascomycetes of common genera to be present. Most species occurred in low frequencies, distributed almost evenly in all treatments. However, in 1999 Microdochium occurred in relatively high frequencies, whereas in the following year no Microdochium species were detected. On the other hand, Fusarium-like organisms were low in 1999, and increased in 2000. Both the DGGE and the sequence analysis revealed that repeated introduction of P. putida WCS358r had no major effects on the ascomycete community in the wheat rhizosphere, but demonstrated a persistent difference between the rhizospheres of potato and wheat.

Plant growth-promoting rhizobacteria can suppress diseases through antagonism between the bacteria and soil-borne pathogens, as well as by inducing a systemic resistance in the plant against both root and foliar pathogens. The generally non-specific character of induced resistance constitutes an increase in the level of basal resistance to several pathogens simultaneously, which is of benefit under natural conditions where multiple pathogens may be present. Specific Pseudomonas strains induce systemic resistance in e.g. carnation, cucumber, radish, tobacco and Arabidopsis, as evidenced by an enhanced defensive capacity upon challenge inoculation. Although some bacterial strains are equally effective in inducing resistance in different plant species, others show specificity, indicating specific recognition between bacteria and plants at the root surface. In carnation, radish and Arabidopsis, the O-antigenic side chain of the bacterial outer membrane lipopolysaccharide acts as an inducing determinant, but other bacterial traits are also involved. Pseudobactin siderophores have been implicated in the induction of resistance in tobacco and Arabidopsis, and another siderophore, pseudomonine, may explain induction of resistance associated with salicylic acid (SA) in radish. Although SA induces phenotypically similar systemic acquired resistance (SAR), it is not necessary for the systemic resistance induced by most rhizobacterial strains. Instead, rhizobacteria-mediated induced systemic resistance (ISR) is dependent on jasmonic acid (JA) and ethylene signaling in the plant. Upon challenge inoculation of induced Arabidopsis plants with a pathogen, leaves expressing SAR exhibit a primed expression of SA-, but not JA/ethylene-responsive defense-related genes, whereas leaves expressing ISR are primed to express JA/ethylene-, but not SA-responsive genes. Combination of ISR and SAR can increase protection against pathogens that are resisted through both pathways, as well as extend protection to a broader spectrum of pathogens than ISR or SAR alone.

To assess effects of plant crop species on rhizosphere ascomycete communities in the field, we compared a wheat monoculture and an alternating crop rotation of wheat and potato. Rhizosphere soil samples were taken at different time points during the growing season in four consecutive years (1999-2002). An ascomycete-specific primer pair (ITS5-ITS4A) was used to amplify internal transcribed spacer (ITS) sequences from total DNA extracts from rhizosphere soil. Amplified DNA was analyzed by denaturing gradient gel electrophoresis (DGGE). Individual bands from DGGE gels were sequenced and compared with known sequences from public databases. DGGE gels representing the ascomycete communities of the continuous wheat and the rotation site were compared and related to ascomycetes identified from the field. The effect of crop rotation exceeded that of the spatial heterogeneity in the field, which was evident after the first year. Significant differences between the ascomycete communities from the rhizospheres of wheat in monoculture and one year after a potato crop were found, indicating a long-term effect of potato. Sequencing of bands excised from the DGGE gels revealed the presence of ascomycetes that are common in agricultural soils.

12 pages, Mutants of Pseudomonas fluorescens WCS374 defective in biosynthesis of the fluorescent siderophore pseudobactin still display siderophore activity, indicating the production of a second siderophore. A recombinant cosmid clone (pMB374-07) of a WCS374 gene library harboring loci necessary for the biosynthesis of salicylic acid (SA) and this second siderophore pseudomonine was isolated. The salicylate biosynthesis region of WCS374 was localized in a 5-kb EcoRI fragment of pMB374-07. The SA and pseudomonine biosynthesis region was identified by transfer of cosmid pMB374-07 to a pseudobactin-deficient strain of P. putida. Sequence analysis of the 5-kb subclone revealed the presence of four open reading frames (ORFs). Products of two ORFs (pmsC and pmsB) showed homologies with chorismate-utilizing enzymes; a third ORF (pmsE) encoded a protein with strong similarity with enzymes involved in the biosynthesis of siderophores in other bacterial species. The region also contained a putative histidine decarboxylase gene (pmsA). A putative promoter region and two predicted iron boxes were localized upstream of pmsC. We determined by reverse transcriptase-mediated PCR that the pmsCEAB genes are cotranscribed and that expression is iron regulated. In vivo expression of SA genes was achieved in P. putida and Escherichia coli cells. In E. coli, deletions affecting the first ORF (pmsC) diminished SA production, whereas deletion of pmsB abolished it completely. The pmsB gene induced low levels of SA production in E. coli when expressed under control of the lacZ promoter. Several lines of evidence indicate that SA and pseudomonine biosynthesis are related. Moreover, we isolated a Tn5 mutant (374-05) that is simultaneously impaired in SA and pseudomonine production., Chemical Institute, University of Copenhagen, Copenhagen,Denmark and Hubrecht Laboratorium, Utrecht, The Netherland

Seventy bacterial isolates from the rhizosphere of tomato were screened for antagonistic activity against the tomato foot and root rot-causing fungal pathogen Fusarium oxysporum f. sp. radicis-lycopersici. One isolate, strain PCL1391, appeared to be an efficient colonizer of tomato roots and an excellent biocontrol strain in an F. oxysporum/tomato test system. Strain PCL1391 was identified as Pseudomonas chlororaphis and further characterization showed that it produces a broad spectrum of antifungal factors (AFFs), including a hydrophobic compound, hydrogen cyanide, chitinase(s), and protease(s). Through mass spectrometry and nuclear magnetic resonance, the hydrophobic compound was identified as phenazine-1-carboxamide (PCN). We have studied the production and action of this AFF both in vitro and in vivo. Using a PCL1391 transposon mutant, with a lux reporter gene inserted in the phenazine biosynthetic operon (phz), we showed that this phenazine biosynthetic mutant was substantially decreased in both in vitro antifungal activity and biocontrol activity. Moreover, with the same mutant it was shown that the phz biosynthetic operon is expressed in the tomato rhizosphere. Comparison of the biocontrol activity of the PCN-producing strain PCL1391 with those of phenazine-1-carboxylic acid (PCA)-producing strains P. fluorescens 2-79 and P. aureofaciens 30-84 showed that the PCN-producing strain is able to suppress disease in the tomato/F. oxysporum system, whereas the PCA-producing strains are not. Comparison of in vitro antifungal activity of PCN and PCA showed that the antifungal activity of PCN was at least 10 times higher at neutral pH, suggesting that this may contribute to the superior biocontrol performance of strain PCL1391 in the tomato/F. oxysporum system.

Pseudobactin production by Pseudomonas putida WCS358 significantly improves biological control of fusarium wilt caused by nonpathogenic Fusarium oxysporum Fo47b10 (P. Lemanceau, P. A. H. M. Bakker, W. J. de Kogel, C. Alabouvette, and B. Schippers, Appl. Environ. Microbiol. 58:2978-2982, 1992). The antagonistic effect of Fo47b10 and purified pseudobactin 358 was studied by using an in vitro bioassay. This bioassay allows studies on interactions among nonpathogenic F. oxysporum Fo47b10, pathogenic F. oxysporum f. sp. dianthi WCS816, and purified pseudobactin 358, the fluorescent siderophore produced by P. putida WCS358. Both nonpathogenic and pathogenic F. oxysporum reduced each other's growth when grown together. However, in these coinoculation experiments, pathogenic F. oxysporum WCS816 was relatively more inhibited in its growth than nonpathogenic F. oxysporum Fo47b10. The antagonism of nonpathogenic F. oxysporum against pathogenic F. oxysporum strongly depends on the ratio of nonpathogenic to pathogenic F. oxysporum densities: the higher this ratio, the stronger the antagonism. This fungal antagonism appears to be mainly associated with the competition for glucose. Pseudobactin 358 reduced the growth of both F. oxysporum strains, whereas ferric pseudobactin 358 did not; antagonism by pseudobactin 358 was then related to competition for iron. However, the pathogenic F. oxysporum strain was more sensitive to this antagonism than the nonpathogenic strain. Pseudobactin 358 reduced the efficiency of glucose metabolism by the fungi. These results suggest that pseudobactin 358 increases the intensity of the antagonism of nonpathogenic F. oxysporum Fo47b10 against pathogenic F. oxysporum WCS816 by making WCS816 more sensitive to the glucose competition by Fo47b10.

Antagonism between fluorescent pseudomonads and plant pathogens is well established as a mechanism of disease suppression. Accordingly, Pseudomonas fluorescens strain WCS417 was demonstrated to suppress Fusarium wilt in a moderately resistant cultivar of carnation, grown in rockwool (Van Peer et al., 1990). Since disease suppression did not occur when a susceptible cultivar was used, it was suggested that mechanisms other than for example iron competition are involved as well. The hypothesis that Pseudomonas fluorescens strain WCS417 can induce resistance was postulated and tested.

Plant growth promotion after treatments with selected strains of fluorescent pseudomonads has been reported for different crops and in different cropping systems. For Pseudomonas putida WCS358 it was demonstrated that increased potato root growth and tuber yields in soil from fields cropped in high frequency to potatoes were due to the production of siderophores by this strain, using Tn5-insertion mutants defective in pseudobactin biosynthesis (Bakker et al., 1990). Suppression of Fusarium wilt of carnation by WCS358 has also been observed; again, mutants defective in siderophore biosynthesis are less effective than the wild type (Duijff and Schippers, unpublished). Lemanceau and Alabouvette (1991) describe a synergistic effect of nonpatho?genic Fusarium spp. and certain strains of fluorescent pseudomonas in the biological control of Fusarium wilt. In combination with the nonpathogenic Fusarium oxysporum strain Fo47, P. putida WCS358 resulted in significantly less Fusarium wilt in carnation, compared to the treatment with Fo47 alone, whereas a mutant of WCS358 defective in siderophore biosynthesis did not enhance biological control by Fo47 (Lemanceau and Bakker, unpublished). These results indicate that P. putida strain WCS358 is especially interesting for biological control with regard to its siderophore biosynthesis. Compe?tition for ferric iron mediated by siderophores, to a large extent depends on the specificity of the siderophores and their receptors. In this study the iron uptake via ferric pseudobactins in strain WCS358 is described. Possibilities to make use of the specificities both for fundamental studies and commercial applications are discussed.

Nonpathogenic Fusarium oxysporum Fo47b10 combined with Pseudomonas putida WCS358 efficiently suppressed fusarium wilt of carnations grown in soilless culture. This suppression was significantly higher than that obtained by inoculation of either antagonistic microorganism alone. The increased suppression obtained by Fo47b10 combined with WCS358 only occurred when Fo47b10 was introduced at a density high enough (at least 10 times higher than that of the pathogen) to be efficient on its own. P. putida WCS358 had no effect on disease severity when inoculated on its own but significantly improved the control achieved with nonpathogenic F. oxysporum Fo47b10. In contrast, a siderophore-negative mutant of WCS358 had no effect on disease severity even in the presence of Fo47b10. Since the densities of both bacterial strains at the root level were similar, the difference between the wild-type WCS358 and the siderophore-negative mutant with regard to the control of fusarium wilt was related to the production of pseudobactin 358. The production of pseudobactin 358 appeared to be responsible for the increased suppression by Fo47b10 combined with WCS358 relative to that with Fo47b10 alone.

Under low iron conditions, Pseudomonas putida WCS358 produces a specific siderophore, pseudobactin 358, and a specific receptor for this siderophore, pupA. Addition of pseudobactin to an agar medium, such that all Fe 3+ in the medium is bound to this siderophore, facilitated selective reisolation of wild-type P. putida WCS358 from soil and potato rhizosphere.

JM3742, a transposon Tn5 derivative of Pseudomonas fluorescens, was deliberately released into the environment as bacterial inoculant of potato seed tubers. The fate of JM3742 in the field was studied and is discussed.

Crop specificity of fluorescent Pseudomonas spp. was studied on potato, wheat, grass, tomato and carnation. Pseudomonas isolates derived from the rhizospheres of these crops were distinquished by comparing their lipopolysaccharide (LPS) and cell envelope protein (CEP) patterns, using sodium dodecyl sulfate polyacrylamide gel electroforesis (SDS-PAGE). The majority of LPS and CEP patterns detected for each crop, were not detected on the other crops. These results suggest a crop specificity for certain Pseudomonas populations. Agglutinins derived from root washings of potato, wheat, grass, tomato and carnation were used to study the involvement of agglutinins in the composition of rhizosphere Pseudomonas populations. Isolates from wheat and grass reacted more frequently with wheat agglutinins compared to isolates from other crops. For the other agglutinins no differential results were obtained with isolates from different crops. The composition of the rhizosphere Pseudomonas populations appeares to differ between the crops studied. For wheat, agglutinins may be involved in this phenomenon.

The use of immunofluorescence colony staining (IFC) for monitoring a strain of Pseudomonas fluorescens , introduced into soil, was studied. The IFC-method was modified and compared to dilution plating on selective media. The results obtained with these two detection methods showed a high degree of correlation. IFC is a relatively fast and quantitative detection method for low populations of P. fluorescens WCS374r in soil.

The world wide interest in Pseudomonas spp. as biocontrol agents is mainly based on their in vitro antagonistic potentials combined with their ability to colonize the roots of many plants efficiently. The properties that confer the colonization abilities on these bacteria are largely unknown. This study focuses on the role of the O-antigenic side chain of the LPS (=lipopolysaccharides) of two plant growth-stimulating Pseudomonas strains, WCS358 and WCS374, in the root colonization process.

During the last few years, Kloepper and coworkers have presented impressive evidence that plant growth-promoting rhizobacteria (PGPR) can protect cucumber plants against fungal, bacterial, and viral diseases. After Wei et al. (14) described induction of systemic resistance of cucumber to Colletotrichum orbiculare by six strains of PGPR, it was repeatedly stated in abstracts of meetings and book chapters that PGPR strains with previously demonstrated induced systemic resistance (ISR) against C. orbiculare led to multiple-pathogen control. However, these strains were not specified, and the communications lacked the technical details necessary to judge whether ISR was the (sole) mechanism responsible. Only recently, two PGPR strains were described as inducing resistance against Fusarium wilt (7), angular leaf spot (8), and anthracnose (9), but the strains had different designations and were not among those used previously (14). Hence, we at first concluded that proof still needed to be provided that the same inducing bacterium could suppress diseases caused by different infectious agents through ISR. Our experiments were conducted before Liu et al. (9) provided details about the strains inducing ISR against C. orbiculare. Nevertheless, we now see that the same PGPR strains did indeed protect against a foliar fungus, a soilborne fungus, and a bacterium, and we admit that we overlooked these recent results when submitting our paper. To conclude that the mechanism responsible for suppression is ISR requires that the disease suppression is shown to be plant mediated and that it extends to plant parts that are not in contact with the inducing agent. This was neatly demonstrated for one PGPR strain in plants protected against Fusarium wilt, because a bioluminescent derivative of strain 89B-27 applied to one part of a split root system did not move to the part inoculated with Fusarium oxysporum f. sp. cucumerinum (7). The other studies did not address this question in detail, although it was mentioned that bacteria inducing systemic resistance were not translocated and did not colonize challenged leaves (6,8). We agree that this implies ISR involvement. However, application of PGPR by seed treatment or cotyledon injection does not exclude colonization of the foliar parts of plants by the bacteria, and hence, microbial antagonism also might play a role. In our studies on ISR in carnation (11), radish (3), and Arabidopsis (10), we have repeatedly shown that inducing bacteria were not recoverable from sites where plants were challenged and that the spatial separation was maintained for the duration of the experiments. Moreover, in all three plant species, ISR was induced by preparations of the bacterial lipopolysaccharide (4,12,13), ruling out any protective effects due to bacterial metabolism. Both Kloepper and coworkers and we have shown that similar strains of PGPR protect various plant species against diseases caused by different types of pathogens, and in doing so, both groups have independently validated the other’s results. PGPR-mediated ISR is an important mechanism of biological disease control, and it can be as effective as pathogen-induced systemic acquired resistance (1,2). Both Leeman et al. (5) and Wei et al. (15) have shown that PGPR are effective in reducing diseases under field conditions. This makes it even more important to further characterize the physiological, biochemical, and molecular mechanisms involved to make optimal use of ISR in plant protection.

A special bioassay on rock wool was used to study the influence of iron availability on the induction of systemic resistance in radish (Raphanus sativus L.) against Fusarium wilt mediated by Pseudomonas fluorescens. In this bioassay, the pathogen (Fusarium oxysporum f. sp. raphani) and a strain of Pseudomonas, salicylic acid (SA), or a pseudobactin were applied at separate locations on the plant root. Strain WCS374 of P. fluorescens and its pseudobactin-minus Tn5 mutant gave greater disease control in the induced systemic resistance bioassay when iron availability in the radish nutrient solution was low than when it was high. Mutants of P. fluorescens strains WCS374 and WCS417 lacking the O-antigenic side chain of the lipopolysaccharide induced resistance at low but not at high iron availability. The purified pseudobactin of strain WCS374, but not the pseudobactins of strains WCS358 and WCS417, induced resistance. Gas chromatography and spectrophotometry were used to detect and measure production of SA by these strains. Strains WCS374 and WCS417 produced 47 and 8 μg of SA per milliliter, respectively, at low iron availability in vitro ; the production of SA decreased rapidly with increasing iron availability. P putida WCS358 did not induce resistance, either at low or at high iron availability, and did not produce SA in vitro. Commercial SA induced resistance at concentrations as low as 100 fg per root. High concentrations (> 1 mg/ml) of SA reduced growth of hyphae from germinated conidia of E oxysporum f. sp. raphani. We hypothesize that the Fe 3+ -chelating SA, produced by selected P. fluorescens strains at low iron availability, is involved in the induction of systemic resistance to Fusarium wilt of radish. The pseudobactin produced by WCS374 may be involved as well. Given these results, it seems appropriate to reevaluate the role of siderophore-mediated competition for iron in the suppression of disease by fluorescent Pseudomonas spp.

In commercial greenhouse trials, Pseudomonas fluorescens strain WCS374 suppressed Fusarium wilt and increased radish yield. In bioassays, the involvement of lipopolysaccharides (LPSs) in induction of systemic resistance was studied. Induction of systemic resistance by selected plant growth-promoting rhizobacteria (PGPR) strains of P. fluorescens was involved in the suppression of Fusarium wilt of radish in a special rockwool bioassay. In this bioassay, the pathogen, Fusarium oxysporum f. sp. raphani, and the PGPR strain were inoculated at spatially separate locations on the plant root and were confined to these locations throughout the experiments. PGPR strains WCS374 and WCS417 of P. fluorescens and their crude cell wall extracts, which contained the LPS or purified LPS (consisting of lipid A/innercore/O-antigen side chain), induced systemic resistance, whereas P putida WCS358 or its crude or purified LPS did not. Neither the phage-resistant mutants of WCS374 and WCS417 lacking the O-antigenic side chain of the LPS nor the crude or purified lipid A/innercore of these mutants reduced disease incidence in this experimental design. Strain WCS374, but not its O-antigen-minus mutant, also induced systemic resistance when applied on the cotyledon of radish on an agar disk cut from a plate culture. The pathogen was delivered on the root in peat 2 days later. Thus, the resistance-inducing O-antigen of strain WCS374 was effective not only onto the root, but also on the cotyledon. In a bioassay with greenhouse soil naturally infested with the Fusarium wilt pathogen of radish, strain WCS374, but not the O-antigen-minus mutant, suppressed disease. This suggests the involvement of induced resistance in natural soil bioassays and commercial greenhouse trials.

Pseudomonas putida strain WCS358, P. fluorescens strain WCS374, and siderophore‐negative mutants of these strains obtained by Tn5 transposon mutagenesis, were used in experiments to assess the role of siderophores in potato growth stimulation by WCS358. The siderophore‐negative mutants used were still able to use the siderophores produced by their respective wild‐types. WCS358 was able to use the siderophore produced by WCS374, but WCS374 could not use the siderophore produced by WCS358. Population densities of siderophore‐negative mutants of WCS358 on potato roots were high in the presence of the wild‐type strains of WCS358 or WCS374, whereas population densities of siderophore‐negative mutants of WCS374 were high only in the presence of wild‐type strain WCS374. It is concluded from these experiments that siderophores of WCS358 and WCS374 are produced in the potato rhizosphere. Root development of potato stem cuttings and potato tuber yields in soils frequently cropped to potato were increased b...

Microorganisms that efficiently colonize plant roots are ideal tools for the production of biocontrol agents, since many major and minor root pathogens are present at the root surface. In the past decade Pseudomonas spp. have received much attention as biocontrol agents (Kloepper et al 1980, Schippers et al 1986). The interest in this group of bacteria is mainly due to their antagonistic potentials in vitro towards pathogens combined with their ability to colonize the roots of many plants efficiently.